Methods of risk assessment and disease classification for appendicitis

ABSTRACT

A method of measuring risk assessment in a patient with appendicitis is disclosed. The method comprises:
         (a) measuring the TRAIL protein level in a blood sample of the patient;   (b) providing a risk assessment based on the TRAIL protein level.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No. PCT/IL2017/051089 having International filing date of Sep. 27, 2017, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 62/401,294 filed on Sep. 29, 2016. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 76882SequenceListing.txt, created on Mar. 26, 2019, comprising 4,425 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods of risk assessment in patients for the management of patient healthcare.

Disease assessment is one of the most important tasks in management of infectious disease patients. Complement to determining infection etiology, predicting patient prognosis may affect various aspects of patient management including treatment, diagnostic tests (e.g., microbiology, blood chemistry, radiology etc), and admission. Timely identification of patients with higher chance for poor prognosis may result in more aggressive patient management procedures including for example, intensive care unit (ICU) admission, advanced therapeutics, invasive diagnostics or surgical intervention, which could reduce complications and mortality.

WO 2013/117746 teaches biomarkers including TNF-related apoptosis-inducing ligand (TRAIL) for distinguishing between a bacterial and viral infection.

Additional background art includes WO 2016/024278.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention, there is provided a method of measuring risk assessment in a patient comprising:

(a) measuring the TRAIL protein level in a blood sample of the patient;

(b) measuring the level of at least one disease-associated parameter in the patient; and

(c) providing a risk assessment based on the combination of the TRAIL protein level and the disease-associated parameter level.

According to an aspect of some embodiments of the present invention, there is provided a method of diagnosing an invasive bacterial infection or a serious bacterial infection (SBI) in a subject comprising measuring the TRAIL protein level in a blood sample of the subject, wherein when the TRAIL protein level is below 25 pg/ml it is indicative of an invasive bacterial infection or SBI.

According to an aspect of some embodiments of the present invention, there is provided a method of determining a treatment course for a subject presenting with an appendicitis comprising measuring the TRAIL protein level in a blood sample of the subject, wherein when the level is below a predetermined amount the subject is recommended for appendectomy.

According to some embodiments of the invention, the measuring the level of at least one disease-associated parameter comprises measuring at least two components of a clinical index of the patient.

According to some embodiments of the invention, the clinical index is selected from the group consisting of APACHE I, APACHE II, APACHE III, CURB-65, SMART-COP, SAPS II, SAPS III, PIM2, CMM, SOFA, MPM, RIFLE, CP, MODS, LODS, Rochester criteria, Philadelphia Criteria, Milwaukee criteria and Ranson score.

According to some embodiments of the invention, the disease associated parameter comprises a disease severity marker.

According to some embodiments of the invention, the disease severity marker is selected from the group consisting of troponin I, creatin, serum albumin, procalcitonin, interleukin-6, C-reactive protein, Pro-adrenomedullin (ProADM), mid-regional ProADM and prostate specific antigen (PSA).

According to some embodiments of the invention, the disease severity marker is selected from the group consisting of troponin I, procalcitonin, Pro-adrenomedullin (ProADM), and prostate specific antigen (PSA).

According to some embodiments of the invention, the patient is a hospitalized patient.

According to some embodiments of the invention, the patient is in the Emergency department.

According to some embodiments of the invention, the patient is in the Intensive care unit (ICU).

According to some embodiments of the invention, the risk measurement is used to determine a management course for the patient, the management course being selected from the group consisting of mechanical ventilation, invasive monitoring, sedation, intensive care admission, surgical intervention, drug of last resort and hospital admittance.

According to some embodiments of the invention, the TRAIL protein level is below 25 pg/ml, the risk assessment is raised.

According to some embodiments of the invention, the level of at least one disease associated parameter is indicative of a low risk patient and the TRAIL protein level is below 25 pg/ml, the patient is classified as an intermediate risk patient.

According to some embodiments of the invention, the level of at least one disease associated parameter is indicative of an intermediate risk patient and the TRAIL protein level is below 25 pg/ml, the patient is classified as a high risk patient.

According to some embodiments of the invention, the level of at least one disease associated parameter is indicative of a high risk patient and the TRAIL protein level is above 25 pg/ml, the patient is classified as an intermediate risk patient.

According to some embodiments of the invention, the predetermined amount is 25 pg/ml.

According to some embodiments of the invention, when the level is above a predetermined amount the subject is recommended for non-surgical treatment.

According to some embodiments of the invention, the non-surgical treatment comprises antibiotic treatment.

According to some embodiments of the invention, the recommendation is further based on at least one measurement selected from the group consisting of white blood cell count (WBC) and C-reactive protein (CRP).

According to some embodiments of the invention, the blood sample is a fraction of whole blood.

According to some embodiments of the invention, the blood sample comprises cells selected from the group consisting of lymphocytes, monocytes and granulocytes.

According to some embodiments of the invention, the fraction is serum or plasma.

According to some embodiments of the invention, the measuring is determined electrophoretically or immunochemically.

According to some embodiments of the invention, the immunochemical determination is effected by flow cytometry, radioimmunoas say, immunofluorescence, lateral flow immunoassay or by an enzyme-linked immunosorbent assay.

According to some embodiments of the invention, the blood sample is a fraction of whole blood.

According to some embodiments of the invention, the fraction is serum or plasma.

According to some embodiments of the invention, the blood sample comprises cells selected from the group consisting of lymphocytes, monocytes and granulocytes.

According to some embodiments of the invention, the invasive bacterial infection is selected from the group consisting of bacteremia, septic arthritis, abdominal abscess, bacterial meningitis, mastoiditis, nephronia, peritonsillar abscess, deep neck infection, periappendicular abscess, and septic shock.

According to some embodiments of the invention, the SBI is selected from the group consisting of bacterial meningitis, bacterial pneumonia, septic arthritis, bacteremia and urinary tract infection (UTI).

According to some embodiments of the invention, the measuring is determined electrophoretically or immunochemically.

According to some embodiments of the invention, the immunochemical determination is effected by lateral flow immunoassay, flow cytometry, radioimmunoassay, immunofluorescence or by an enzyme-linked immunosorbent assay.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1: Clinical study workflow.

FIG. 2: Distribution of age of the patients enrolled in the clinical study.

FIG. 3: Distribution of physiological systems of the infectious disease patients enrolled in the clinical study.

FIG. 4: Distribution of clinical syndromes of the infectious disease patients enrolled in the clinical study.

FIG. 5: Distribution of maximal body temperatures of the infectious disease patients enrolled in the clinical study.

FIG. 6: Distribution of time from initiation of symptoms of the infectious disease patients enrolled in the clinical study.

FIG. 7: Distribution of comorbidities of the patients enrolled in the clinical study.

FIG. 8: Selected pathogens isolated from infectious disease patients enrolled in the clinical study.

FIG. 9: Distribution of TRAIL serum levels in patients with different infection types. Box plots for TRAIL measured over the study cohort are presented. Line and circle inside the rectangle correspond to group median and average respectively; t-test p-values between bacterial and viral groups and between infectious (bacterial and viral) vs. non-infectious (including healthy subjects) are depicted.

FIG. 10: Different biomarker cutoffs indicative of different clinical states. (A) Highly sensitive CRP (HCRP; 0-10 μg/ml) is used for risk stratification of patients with suspicious of myocardial infarction, while higher levels of CRP (>10 μg/ml) are indicative of inflammatory states, including bacterial and viral infections. (B) Highly sensitive TRAIL (HTRAIL; 0.5-25 pg/ml) serves as an indicator for disease severity, while higher levels of TRAIL serves as indicators for bacterial infection (25-70 pg/ml), healthy state (70-90 pg/ml), or viral infections (90-3000 pg/ml).

FIG. 11: Distribution of study patients according to different serum TRAIL levels in the studied cohort.

FIG. 12: Average hospitalization duration (days) in patients with different TRAIL levels.

FIG. 13: Intensive care unit (ICU) admission rates in infectious patients with different TRAIL levels.

FIG. 14: Serum TRAIL levels are lower in patients admitted to the ICU

FIG. 15: Patients with TRAIL levels <25 pg/ml (an exemplary cutoff) are statistically enriched in sub-group of patients with severe clinical syndromes. p-values were calculated using the hyper-geometric distribution. In the entire cohort 93 out of 653 patients (14%) had TRAIL<25 pg/ml. 7 out of 11 (64%, p<10⁻³) patients with bacteremia had TRAIL<25 pg/ml, and all 7 patients with septic shock (100%, p<10⁻⁶) had TRAIL<25 pg/m.

FIGS. 16A-C: Exemplary algorithm for combining TRAIL/HTRAIL with established methods for risk assessment as indicated (A—with initial clinical judgment; B—with a generic severity of disease classification score; C—with the APACHE II score). Adding the TRAIL/HTRAIL data can alter the risk level of a patient leading to improved patient management and health outcome.

FIG. 17: Exemplary algorithm for combining different levels of TRAIL with established methods for risk assessment to alter the risk level of a patient leading to improved patient management and health outcome.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods of risk assessment in patients for the management of patient healthcare. The risk assessment is based on the level of serum protein TNF-related apoptosis-inducing ligand (TRAIL) levels.

Disease assessment is one of the most important tasks in management of infectious disease patients. As a complement to determining infection etiology, predicting patient prognosis may affect various aspects of patient management including treatment, diagnostic tests (e.g., microbiology, blood chemistry, radiology etc), and admission. Timely identification of patients with higher chance for poor prognosis may result in more aggressive patient management procedures including for example, ICU admission, advanced therapeutics, invasive diagnostics or surgical intervention, which could reduce complications and mortality. The present inventors previously discovered that TNF-related apoptosis-inducing ligand (TRAIL) levels are decreased in bacterial patients and increased in viral patients compared to non-infectious subjects (FIG. 9). Based on their findings, they suggested TRAIL as a diagnostic marker for distinguishing between bacterial and viral patients (e.g. WO 2013/117746). Whilst reducing the present invention to practice, the present inventors have now noted that very low TRAIL levels are correlated with different aspects of disease severity and thus could be used for tailoring the correct patient management course. More specifically, the present inventors have shown that very low TRAIL levels are correlated with higher rates of ICU admission (FIGS. 13 and 14) and longer hospital length of stay (FIG. 12). The present inventors propose that the serum TRAIL level can be used to measure risk assessment in patients so as to ultimately provide a higher healthcare standard for the patient. The level of TRAIL may be used to assess the risk to the patient of a pre-diagnosed disease. Furthermore, the present inventors propose that the serum TRAIL level can be used as a prognostic indicator in combination with additional disease severity markers to provide a more accurate and dependable risk assessment for the patient—see for example FIGS. 16A-C.

Thus, according to a first aspect of the present invention there is provided a method of measuring risk assessment in a patient comprising:

(a) measuring the TRAIL protein level in a blood sample of the patient;

(b) measuring the level of at least one disease-associated parameter in the patient; and

(c) providing a risk assessment based on the TRAIL protein level and disease-associated parameter level.

The term “risk assessment” refers to as assignment of a probability to experience certain adverse events (e.g. death, hospitalization or admission to ICU) to an individual. Hereby, the individual may preferably be accounted to a certain risk category, wherein categories comprise for instance high risk versus low risk, or risk categories based on numeral values, such as risk category 1, 2, 3, etc.

According to a specific embodiment, the TRAIL protein level is used to stratify the subject into one of four levels—low, intermediate, high and critical.

In one embodiment, the risk assessment is made in the emergency department of a hospital.

Emergency departments (ED) are progressively overwhelmed by patients with both urgent and non-urgent problems. This leads to overfilled ED waiting rooms with long waiting times, detrimental outcomes and unsatisfied patients. As a result, patients needing urgent care may not be treated in time, whereas patients with non-urgent problems may unnecessarily receive expensive and dispensable treatments. Time to effective treatment is among the key predictors for outcomes across different medical conditions, including patients with septicaemia, pneumonia, stroke and myocardial infarction. For these reasons, the present inventors propose use of the presently disclosed risk stratification system in the ED is essential for an optimal initial triage of medical patients.

In another embodiment, the risk assessment is made in the intensive care unit of a hospital.

The risk measurement may be used to determine a management course for the patient. The risk measurement may aid in selection of treatment priority and also site-of-care decisions (i.e. outpatient vs. inpatient management) and early identification and organization of post-acute care needs.

When a patient has been assessed as being at high risk, the management course is typically more aggressive than if he had not been assessed as being at high risk. Thus, treatment options such as mechanical ventilation, life support, catheterization, hemofiltration, invasive monitoring, sedation, intensive care admission, surgical intervention, drug of last resort and hospital admittance may be selected which may otherwise not have been considered the preferred method of treatment if the patient had not been assessed as being at high risk.

Classification of subjects into subgroups as performed in aspects of the present invention is preferably done with an acceptable level of clinical or diagnostic accuracy. An “acceptable degree of diagnostic accuracy”, is herein defined as a test or assay (such as the test used in some aspects of the invention) in which the AUC (area under the ROC curve for the test or assay) is at least 0.60, desirably at least 0.65, more desirably at least 0.70, preferably at least 0.75, more preferably at least 0.80, and most preferably at least 0.85.

By a “very high degree of diagnostic accuracy”, it is meant a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.75, 0.80, desirably at least 0.85, more desirably at least 0.875, preferably at least 0.90, more preferably at least 0.925, and most preferably at least 0.95.

Alternatively, the methods predict risk with at least 75% total accuracy, more preferably 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater total accuracy. Alternatively, the methods predict the correct management or treatment with an MCC larger than 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0.

A “subject” in the context of the present invention is typically a mammal (e.g. human, dog, cat, horse, cow, sheep, pig or goat). According to another embodiment, the subject is a bird (e.g. chicken, turkey, duck or goose). According to a particular embodiment, the subject is a human. The subject may be male or female. The subject may be an adult (e.g. older than 18, 21, or 22 years or a child (e.g. younger than 18, 21 or 22 years). In another embodiment, the subject is an adolescent (between 12 and 21 years), an infant (29 days to less than 2 years of age) or a neonate (birth through the first 28 days of life).

The subjects of this aspect of the present invention typically present with symptoms of a disease—i.e. the subject is a patient.

Exemplary symptoms of a disease include but are not limited to abnormal blood pressure, abnormal heart rate, abnormal red blood count, abnormal white blood count, abnormal body temperature, abnormal respiratory rate, abnormal lucidity or alertness.

In one embodiment the symptoms which the subject may present with are symptoms of an infectious disease. Exemplary symptoms include but are not limited to fever, nausea, headache, sore throat, runny nose, rash and/or muscle soreness.

In another embodiment the symptoms which the subject may present with are symptoms of a cardiac disease (e.g. chest pain, high blood pressure).

According to a particular embodiment, the subject does not show signs of having had a heart attack (e.g. has a normal level of creatine kinase, troponin or serum myoglobin, and/or has a normal ECG or EKG).

According to another embodiment, the subject does not have cancer.

In one embodiment, the subject is known not to have sepsis.

In still another embodiment, the symptoms which the subject may present with are symptoms of a pulmonary disease (e.g. cough, breathing difficulty).

In still another embodiment, the symptoms which the subject may present with are symptoms of a metabolic disease (e.g. high blood sugar).

It will be appreciated that the subject may have a known pre-diagnosed disease. Exemplary disease include bacterial infections (e.g. bacteremia, meningitis, respiratory tract infections, urinal tract infections etc.), viral infections, COPD, chronic lung disease, diabetes, hypertension, sepsis, physical injury and trauma, cardiovascular diseases, multi-organ failure associated diseases, drug-induced nephrotoxicity, acute kidney disease, renal injury, kidney failure, advanced cirrhosis and liver failure, acute or chronic left heart failure, pulmonary hypertension with/without right heart failure, and various types of malignancies.

Sepsis is a life-threatening condition that is caused by inflammatory response to an infection. The early diagnosis of sepsis is essential for clinical intervention before the disease rapidly progresses beyond initial stages to the more severe stages, such as severe sepsis or septic shock, which are associated with high mortality.

According to one embodiment, sepsis may be diagnosed as the presence of SIRS criteria in the presence of a known infection. (SIRS may be defined as 2 or more of the following variables: fever of more than 38° C. (100.4° F.) or less than 36° C. (96.8° F.); heart rate of more than 90 beats per minute; respiratory rate of more than 20 breaths per minute or arterial carbon dioxide tension (PaCO2) of less than 32 mm Hg; abnormal white blood cell count (>12,000/μL or <4,000/μL or >10% immature [band] forms)).

In another embodiment, sepsis is diagnosed in a subject suspected of having an infection and which fulfils each of the three criteria:

Respiratory rate greater or equal to 22/min;

Altered mentation (e.g. a Glasgow coma score of less than 15);

Systolic blood pressure lower than or equal to 100 mmHg.

Further criteria for diagnosing sepsis are disclosed in Singer et al. 2016, 315(8):801-810 JAMA.

Chronic obstructive pulmonary disease (COPD) is an obstructive, inflammatory lung disease characterized by long-term poor airflow. The main symptoms include shortness of breath and cough with sputum production. COPD is a progressive disease, worsening over time.

For any of the aspects disclosed herein, the term “measuring” or “measurement,” or alternatively “detecting” or “detection”, means assessing the presence, absence, quantity or amount (which can be an effective amount) of the determinant within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such determinants.

The phrase “disease associated parameter” includes biomarkers or determinants such as polypeptides, polynucleotides and metabolites as well as clinical determinants.

Examples of clinical determinants include but are not limited to ANC, ALC, Neu (%), Lym (%), Mono (%), Maximal temperature, time from symptoms, heart rate, blood pressure, age, Creatinine (Cr), Potassium (K), Pulse and Urea.

Preferably, the disease associated parameter is a biomarker that correlates with risk to the patient (i.e. a disease severity marker).

These include, for example proadrenomedullin (ADM), mid-regional proadrenomedullin (MR-proADM), a stable peptide of the precursor of ADM, C-reactive protein (CRP) and procalcitonin (PCT) which are used as markers of infection/vasodilation; high-sensitivity troponin T assay and natriuretic peptides which are used as cardiac dysfunction markers; copeptin and cortisol which are used as markers of stress; plasma neutrophil gelatinase-associated lipocalin, the soluble form of urokinase-type plasminogen activator and urea which are used as markers of kidney dysfunction; thyroid hormones and proEndothelin-1 which are used as a marker of endothelial activation and lactate which is used as a marker of organ dysfunction. According to a particular embodiment, the disease severity marker is selected from the group consisting of the level of troponin I, procalcitonin, Pro-adrenomedullin (ProADM), Mid-region pro-adrenomedullin (MR-proADM) and prostate specific antigen (PSA).

According to a particular embodiment, the disease associated parameter is Mid-region pro-adrenomedullin (MR-proADM) or Pro-adrenomedullin (ProADM).

Other exemplary blood biomarkers which may be measured according to this aspect of the present invention include but are not limited to creatin, serum albumin, and interleukin-6.

In a particular embodiment, measuring the level of at least one disease-associated parameter comprises measuring at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least nine parameters of a clinical index of the subject and providing a risk score based on the clinical index.

In one embodiment, measuring the level of at least one disease-associated parameter comprises measuring all the parameters of a clinical index of the subject.

Exemplary clinical indices include but are not limited to Acute Physiology and Chronic Health Evaluation (APACHE II) as a measure of how likely to make it out of intensive care unit; Simplified Acute Physiology (SAP) score; Glasgow Coma Score (GCS) as an assessment of consciousness; Sequential Organ Failure Assessment (SOFA) score as an assessment of person's organ function or rate of failure; Apgar Assessment of a newborn's adjustment to life; Pain perception profile; visual analogue scale (VAS); quality of life metrics such as EDLQ, SF36; depression scale such as CES-D; impact of event scale (IES); or thrombosis risk assessment, or trend therein, or combination of above.

According to a particular embodiment, the clinical index is Acute Physiology and Chronic Health Evaluation II (APACHE II). This system is an example of a severity of disease classification system that uses a point score based upon initial values of 12 routine physiologic measurements that include: temperature, mean arterial pressure, pH arterial, heart rate, respiratory rate, AaDO2 or PaO2, sodium, potassium, creatinine, hematocrit, white blood cell count, and Glasgow Coma Scale. These parameters are measured during the first 24 hours after admission, and utilized in additional to information about previous health status (recent surgery, history of severe organ insufficiency, immunocompromised state) and baseline demographics such as age. An integer score from 0 to 71 is calculated wherein higher scores correspond to more severe disease and a higher risk of death.

Many other predictive models have been developed for various purposes which are contemplated by the present invention. Such predictive models are used for determining population-based outcome risks. By way of illustration and not as a limitation, a partial list of predictive models comprises SAPS II expanded and predicted mortality, SAPS II and predicted mortality, APACHE I-IV and predicted mortality, SOFA (Sequential Organ Failure Assessment), MODS (Multiple Organ Dysfunction Score), ODIN (Organ Dysfunctions and/or Infection), MPM (Mortality Probability Model), MPM II LODS (Logistic Organ Dysfunction System), TRIOS (Three days Recalibrated ICU Outcome Score), EUROSCORE (cardiac surgery), ONTARIO (cardiac surgery), Parsonnet score (cardiac-surgery), System 97 score (cardiac surgery), QMMI score (coronary surgery), Early mortality risk in redocoronary artery surgery, MPM for cancer patients, POSSUM (Physiologic and Operative Severity Score for the enUmeration of Mortality and Morbidity) (surgery, any), Portsmouth POSSUM (surgery, any), IRISS score: graft failure after lung transplantation, Glasgow Coma Score, ISS (Injury Severity Score), RTS (Revised Trauma Score), TRISS (Trauma Injury Severity Score), ASCOT (A Severity Characterization Of Trauma), 24 h-ICU Trauma Score, TISS (Therapeutic Intervention Scoring System), TISS-28 (simplified TISS), PRISM (Pediatric RISk of Mortality), P-MODS (Pediatric Multiple Organ Dysfunction Score), DORA (Dynamic Objective Risk Assessment), PELOD (Pediatric Logistic Organ Dysfunction), PIM II (Paediatric Index of Mortality II), PIM (Paediatric Index of Mortality), CRIB II (Clinical Risk Index for Babies), CRIB (Clinical Risk Index for Babies), SNAP (Score for Neonatal Acute Physiology), SNAP-PE (SNAP Perinatal Extension), SNAP II and SNAPPE II, MSSS (Meningococcal Septic Shock Score), GMSPS (Glasgow Meningococcal Septicaemia Prognostic Score), Rotterdam Score (meningococcal septic shock), Children's Coma Score (Raimondi), Paediatric Coma Scale (Simpson & Reilly), and Pediatric Trauma Score, Rochester criteria, Philadelphia Criteria, Milwaukee criteria, the last three being specific to neonatal fever/sepsis. Of course, the above list of quality of care metrics directed to health risk to the patient is not limiting, and other miscellaneous scores and assessments known in the medical field can be used.

Depending on the nature of the disease associated parameter, the measurement may be made to the body per se (e.g. blood pressure, temperature) or may be made in a sample retrieved from the patient.

A “sample” in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, whole blood, serum, plasma, saliva, mucus, breath, urine, CSF, sputum, sweat, stool, hair, seminal fluid, biopsy, rhinorrhea, tissue biopsy, cytological sample, platelets, reticulocytes, leukocytes, epithelial cells, or whole blood cells.

In a particular embodiment, the sample is a blood sample—e.g. serum, plasma, whole blood. The sample may be a venous sample, peripheral blood mononuclear cell sample or a peripheral blood sample. Preferably, the sample comprises white blood cells including for example granulocytes, lymphocytes and/or monocytes. In one embodiment, the sample is depleted of red blood cells.

The sample is preferably derived from the subject no more than 72 hours, no more than 60 hours, no more than 48 hours, no more than 36 hours, no more than one 24 hours or even no more than 12 hours following symptom onset.

The sample may be fresh or frozen.

Trail:

The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. The present invention contemplates measuring either the soluble and/or the membrane form of this protein. In one embodiment, only the soluble form of this protein is measured. Additional names of the gene include without limitations APO2L, TNF-related apoptosis-inducing ligand, TNFSF10 and CD253. This protein binds to several members of the TNF receptor superfamily such as TNFRSF10A/TRAILR1, TNFRSF10B/TRAILR2, TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4, and possibly also to TNFRSF11B/OPG.

Additional information concerning TRAIL is provided in Table 1, herein below.

TABLE 1 Protein RefSeq DNA symbol Full Gene Name sequence RefSeq proteins TRAIL Tumor necrosis factor NC_000003.12 NP_001177871.1 superfamily member NC_018914.2 NP_001177872.1 10 NT_005612.17 NP_003801.1

Exemplary amino acid sequences of TRAIL are set forth in SEQ ID NOs. 1-3.

Methods of measuring the level of TRAIL polypeptide are well known in the art and include, e.g., immunoassays based on antibodies to proteins, aptamers or molecular imprints.

TRAIL can be detected in any suitable manner, but are typically detected by contacting a sample from the subject with an antibody, which binds the TRAIL and then detecting the presence or absence of a reaction product. The antibody may be monoclonal, polyclonal, chimeric, or a fragment of the foregoing, and the step of detecting the reaction product may be carried out with any suitable immunoassay.

In one embodiment, the antibody which specifically binds the determinant is attached (either directly or indirectly) to a signal producing label, including but not limited to a radioactive label, an enzymatic label, a hapten, a reporter dye or a fluorescent label.

Immunoassays carried out in accordance with some embodiments of the present invention may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves the specific antibody (e.g., anti-determinant antibody), a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof can be carried out in a homogeneous solution. Immunochemical labels, which may be employed, include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes.

In a heterogeneous assay approach, the reagents are usually the sample, the antibody, and means for producing a detectable signal. Samples as described above may be used. The antibody can be immobilized on a support, such as a bead (such as protein A and protein G agarose beads), plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the sample. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, or enzyme labels. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are oligonucleotides, immunoblotting, immunofluorescence methods, immunoprecipitation, chemiluminescence methods, electrochemiluminescence (ECL) or enzyme-linked immunoassays.

Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see also U.S. Pat. No. 4,727,022 to Skold et al., titled “Methods for Modulating Ligand-Receptor Interactions and their Application,” U.S. Pat. No. 4,659,678 to Forrest et al., titled “Immunoassay of Antigens,” U.S. Pat. No. 4,376,110 to David et al., titled “Immunometric Assays Using Monoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled “Macromolecular Environment Control in Specific Receptor Assays,” U.S. Pat. No. 4,233,402 to Maggio et al., titled “Reagents and Method Employing Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al., titled “Heterogenous Specific Binding Assay Employing a Coenzyme as Label.” The determinant can also be detected with antibodies using flow cytometry. Those skilled in the art will be familiar with flow cytometric techniques which may be useful in carrying out the methods disclosed herein (Shapiro 2005). These include, without limitation, Cytokine Bead Array (Becton Dickinson) and Luminex technology.

Antibodies can be conjugated to a solid support suitable for a diagnostic assay (e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as passive binding. Antibodies as described herein may likewise be conjugated to detectable labels or groups such as radiolabels (e.g., ³⁵S, ¹²⁵I ¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein, Alexa, green fluorescent protein, rhodamine) in accordance with known techniques.

In particular embodiments, the antibodies of the present invention are monoclonal antibodies.

Suitable sources for antibodies for the detection of determinants include commercially available sources such as, for example, Abazyme, Abnova, AssayPro, Affinity Biologicals, AntibodyShop, Aviva bioscience, Biogenesis, Biosense Laboratories, Calbiochem, Cell Sciences, Chemicon International, Chemokine, Clontech, Cytolab, DAKO, Diagnostic BioSystems, eBioscience, Endocrine Technologies, Enzo Biochem, Eurogentec, Fusion Antibodies, Genesis Biotech, GloboZymes, Haematologic Technologies, Immunodetect, Immunodiagnostik, Immunometrics, Immunostar, Immunovision, Biogenex, Invitrogen, Jackson ImmunoResearch Laboratory, KMI Diagnostics, Koma Biotech, LabFrontier Life Science Institute, Lee Laboratories, Lifescreen, Maine Biotechnology Services, Mediclone, MicroPharm Ltd., ModiQuest, Molecular Innovations, Molecular Probes, Neoclone, Neuromics, New England Biolabs, Novocastra, Novus Biologicals, Oncogene Research Products, Orbigen, Oxford Biotechnology, Panvera, PerkinElmer Life Sciences, Pharmingen, Phoenix Pharmaceuticals, Pierce Chemical Company, Polymun Scientific, Polysiences, Inc., Promega Corporation, Proteogenix, Protos Immunoresearch, QED Biosciences, Inc., R&D Systems, Repligen, Research Diagnostics, Roboscreen, Santa Cruz Biotechnology, Seikagaku America, Serological Corporation, Serotec, SigmaAldrich, StemCell Technologies, Synaptic Systems GmbH, Technopharm, Terra Nova Biotechnology, TiterMax, Trillium Diagnostics, Upstate Biotechnology, US Biological, Vector Laboratories, Wako Pure Chemical Industries, and Zeptometrix. However, the skilled artisan can routinely make antibodies, against any of the polypeptide determinants described herein.

The presence of a label can be detected by inspection, or a detector which monitors a particular probe or probe combination is used to detect the detection reagent label. Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Those skilled in the art will be familiar with numerous suitable detectors that widely available from a variety of commercial sources and may be useful for carrying out the method disclosed herein. Commonly, an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis. See generally The Immunoassay Handbook [The Immunoassay Handbook. Third Edition. 2005].

Examples of “Monoclonal antibodies for measuring TRAIL”, include without limitation: Mouse, Monoclonal (55B709-3) IgG; Mouse, Monoclonal (2E5) IgG1; Mouse, Monoclonal (2E05) IgG1; Mouse, Monoclonal (M912292) IgG1 kappa; Mouse, Monoclonal (IIIF6) IgG2b; Mouse, Monoclonal (2E1-1B9) IgG1; Mouse, Monoclonal (RIK-2) IgG1, kappa; Mouse, Monoclonal M181 IgG1; Mouse, Monoclonal V110E IgG2b; Mouse, Monoclonal MAB375 IgG1; Mouse, Monoclonal MAB687 IgG1; Mouse, Monoclonal HS501 IgG1; Mouse, Monoclonal clone 75411.11 Mouse IgG1; Mouse, Monoclonal T8175-50 IgG; Mouse, Monoclonal 2B2.108 IgG1; Mouse, Monoclonal B-T24 IgG1; Mouse, Monoclonal 55B709.3 IgG1; Mouse, Monoclonal D3 IgG1; Goat, Monoclonal C19 IgG; Rabbit, Monoclonal H257 IgG; Mouse, Monoclonal 500-M49 IgG; Mouse, Monoclonal 05-607 IgG; Mouse, Monoclonal B-T24 IgG1; Rat, Monoclonal (N2B2), IgG2a, kappa; Mouse, Monoclonal (1A7-2B7), IgG1; Mouse, Monoclonal (55B709.3), IgG and Mouse, Monoclonal B-S23*IgG1.

Soluble TRAIL and membrane TRAIL can be distinguished by using different measuring techniques and samples. For example, Soluble TRAL can be measured without limitation in cell free samples such as serum or plasma, using without limitation lateral flow immunoassay (LFIA), as further described herein below. Membrane TRAIL can be measured in samples that contain cells using cell based assays including without limitation flow cytometry, ELISA, and other immunoassays.

Lateral Flow Immunoassays (LFIA):

This is a technology which allows rapid measurement of analytes at the point of care (POC) and its underlying principles are described below. According to one embodiment, LFIA is used in the context of a hand-held device.

The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third ‘capture’ molecule binds the complex.

After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones the fluid enters the final porous material, the wick, that simply acts as a waste container. Lateral Flow Tests can operate as either competitive or sandwich assays.

Different formats may be adopted in LFIA. Strips used for LFIA contain four main components. A brief description of each is given before describing format types.

Sample Application Pad:

It is made of cellulose and/or glass fiber and sample is applied on this pad to start assay. Its function is to transport the sample to other components of lateral flow test strip (LFTS). Sample pad should be capable of transportation of the sample in a smooth, continuous and homogenous manner. Sample application pads are sometimes designed to pretreat the sample before its transportation. This pretreatment may include separation of sample components, removal of interferences, adjustment of pH, etc.

Conjugate Pad:

It is the place where labeled biorecognition molecules are dispensed. Material of conjugate pad should immediately release labeled conjugate upon contact with moving liquid sample. Labeled conjugate should stay stable over entire life span of lateral flow strip. Any variations in dispensing, drying or release of conjugate can change results of assay significantly. Poor preparation of labeled conjugate can adversely affect sensitivity of assay. Glass fiber, cellulose, polyesters and some other materials are used to make conjugate pad for LFIA. Nature of conjugate pad material has an effect on release of labeled conjugate and sensitivity of assay.

Nitrocellulose Membrane:

It is highly critical in determining sensitivity of LFIA. Nitrocellulose membranes are available in different grades. Test and control lines are drawn over this piece of membrane. So an ideal membrane should provide support and good binding to capture probes (antibodies, aptamers etc.). Nonspecific adsorption over test and control lines may affect results of assay significantly, thus a good membrane will be characterized by lesser non-specific adsorption in the regions of test and control lines. Wicking rate of nitrocellulose membrane can influence assay sensitivity. These membranes are easy to use, inexpensive, and offer high affinity for proteins and other biomolecules. Proper dispensing of bioreagents, drying and blocking play a role in improving sensitivity of assay.

Adsorbent Pad:

It works as sink at the end of the strip. It also helps in maintaining flow rate of the liquid over the membrane and stops back flow of the sample. Adsorbent capacity to hold liquid can play an important role in results of assay.

All these components are fixed or mounted over a backing card. Materials for backing card are highly flexible because they have nothing to do with LFIA except providing a platform for proper assembling of all the components. Thus backing card serves as a support and it makes easy to handle the strip.

Major steps in LFIA are (i) preparation of antibody against target analyte (ii) preparation of label (iii) labeling of biorecognition molecules (iv) assembling of all components onto a backing card after dispensing of reagents at their proper pads (v) application of sample and obtaining results.

Sandwich Format:

In a typical format, label (Enzymes or nanoparticles or fluorescence dyes) coated antibody or aptamer is immobilized at conjugate pad. This is a temporary adsorption which can be flushed away by flow of any buffer solution. A primary antibody or aptamer against target analyte is immobilized over test line. A secondary antibody or probe against labeled conjugate antibody/aptamer is immobilized at control zone.

Sample containing the analyte is applied to the sample application pad and it subsequently migrates to the other parts of strip. At conjugate pad, target analyte is captured by the immobilized labeled antibody or aptamer conjugate and results in the formation of labeled antibody conjugate/analyte complex. This complex now reaches at nitrocellulose membrane and moves under capillary action. At test line, label antibody conjugate/analyte complex is captured by another antibody which is primary to the analyte. Analyte becomes sandwiched between labeled and primary antibodies forming labeled antibody conjugate/analyte/primary antibody complex. Excess labeled antibody conjugate will be captured at control zone by secondary antibody. Buffer or excess solution goes to absorption pad. Intensity of color at test line corresponds to the amount of target analyte and is measured with an optical strip reader or visually inspected. Appearance of color at control line ensures that a strip is functioning properly.

Competitive Format:

Such a format suits best for low molecular weight compounds which cannot bind two antibodies simultaneously. Absence of color at test line is an indication for the presence of analyte while appearance of color both at test and control lines indicates a negative result. Competitive format has two layouts. In the first layout, solution containing target analyte is applied onto the sample application pad and prefixed labeled biomolecule (antibody/aptamer) conjugate gets hydrated and starts flowing with moving liquid. Test line contains pre-immobilized antigen (same analyte to be detected) which binds specifically to label conjugate. Control line contains pre-immobilized secondary antibody which has the ability to bind with labeled antibody conjugate. When liquid sample reaches at the test line, pre-immobilized antigen will bind to the labeled conjugate in case target analyte in sample solution is absent or present in such a low quantity that some sites of labeled antibody conjugate were vacant. Antigen in the sample solution and the one which is immobilized at test line of strip compete to bind with labeled conjugate. In another layout, labeled analyte conjugate is dispensed at conjugate pad while a primary antibody to analyte is dispensed at test line. After application of analyte solution a competition takes place between analyte and labeled analyte to bind with primary antibody at test line.

Multiplex Detection Format:

Multiplex detection format is used for detection of more than one target species and assay is performed over the strip containing test lines equal to number of target species to be analyzed. It is highly desirable to analyze multiple analytes simultaneously under same set of conditions. Multiplex detection format is very useful in clinical diagnosis where multiple analytes which are inter-dependent in deciding about the stage of a disease are to be detected. Lateral flow strips for this purpose can be built in various ways i.e. by increasing length and test lines on conventional strip, making other structures like stars or T-shapes. Shape of strip for LFIA will be dictated by number of target analytes. Miniaturized versions of LFIA based on microarrays for multiplex detection of DNA sequences have been reported to have several advantages such as less consumption of test reagents, requirement of lesser sample volume and better sensitivity.

Labels:

Any material that is used as a label should be detectable at very low concentrations and it should retain its properties upon conjugation with biorecognition molecules. This conjugation is also expected not to change features of biorecognition probes. Ease in conjugation with biomolecules and stability over longer period of time are desirable features for a good label. Concentrations of labels down to 10⁻⁹ M are optically detectable. After the completion of assay, some labels generate direct signal (as color from gold colloidal) while others require additional steps to produce analytical signal (as enzymes produce detectable product upon reaction with suitable substrate). Hence the labels which give direct signal are preferable in LFA because of less time consumption and reduced procedure.

Gold Nanoparticles:

Colloidal gold nanoparticles are the most commonly used labels in LFA. Colloidal gold is inert and gives very perfect spherical particles. These particles have very high affinity toward biomolecules and can be easily functionalized. Optical properties of gold nanoparticles are dependent on size and shape. Size of particles can be tuned by use of suitable chemical additives. Their unique features include environment friendly preparation, high affinity toward proteins and biomolecules, enhanced stability, exceptionally higher values for charge transfer and good optical signaling. Optical signal of gold nanoparticles in colorimetric LFA can be amplified by deposition of silver, gold nanoparticles and enzymes.

Magnetic Particles and Aggregates:

Colored magnetic particles produce color at the test line which is measured by an optical strip reader but magnetic signals coming from magnetic particles can also be used as detection signals and recorded by a magnetic assay reader. Magnetic signals are stable for longer time compared to optical signals and they enhance sensitivity of LFA by 10 to 1000 folds.

Fluorescent and Luminescent Materials:

Fluorescent molecules are widely used in LFA as labels and the amount of fluorescence is used to quantitate the concentration of analyte in the sample. Detection of proteins is accomplished by using organic fluorophores such as rhodamine as labels in LFA.

Current developments in nanomaterial have headed to manufacture of quantum dots which display very unique electrical and optical properties. These semiconducting particles are not only water soluble but can also be easily combined with biomolecules because of closeness in dimensions. Owing to their unique optical properties, quantum dots have come up as a substitute to organic fluorescent dyes Like gold nanoparticles QDs show size dependent optical properties and a broad spectrum of wavelengths can be monitored. Single light source is sufficient to excite quantum dots of all different sizes. QDs have high photo stability and absorption coefficients.

Upconverting phosphors (UCP) are characterized by their excitation in infra-red region and emission in high energy visible region. Compared to other fluorescent materials, they have a unique advantage of not showing any auto fluorescence. Because of their excitation in IR regions, they do not photo degrade biomolecules. A major advantage lies in their production from easily available bulk materials. Although difference in batch to batch preparation of UCP reporters can affect sensitivity of analysis in LFA, it was observed that they can enhance sensitivity of analytical signal by 10 to 100 folds compared to gold nanoparticles or colored latex beads, when analysis is carried out under same set of biological conditions.

Enzymes:

Enzymes are also employed as labels in LFA. But they increase one step in LFA which is application of suitable substrate after complete assay. This substrate will produce color at test and control lines as a result of enzymatic reaction. In case of enzymes, selection of suitable enzyme substrate combination is one necessary requirement in order to get a colored product for strip reader or electroactive product for electrochemical detection. In other words, sensitivity of detection is dependent on enzyme substrate combination.

Colloidal Carbon:

Colloidal carbon is comparatively inexpensive label and its production can be easily scaled up. Because of their black color, carbon NPs can be easily detected with high sensitivity. Colloidal carbon can be functionalized with a large variety of biomolecules for detection of low and high molecular weight analytes.

Detection Systems:

In case of gold nanoparticles or other color producing labels, qualitative or semi-quantitative analysis can be done by visual inspection of colors at test and control lines. The major advantage of visual inspection is rapid qualitative answer in “Yes” or “NO”. Such quick replies about presence of an analyte in clinical analysis have very high importance. Such tests help doctors to make an immediate decision near the patients in hospitals in situations where test results from central labs cannot be waited for because of huge time consumption. But for quantification, optical strip readers are employed for measurement of the intensity of colors produced at test and control lines of strip. This is achieved by inserting the strips into a strip reader and intensities are recorded simultaneously by imaging softwares. Optical images of the strips can also be recorded with a camera and then processed by using a suitable software. Procedure includes proper placement of strip under the camera and a controlled amount of light is thrown on the areas to be observed. Such systems use monochromatic light and wavelength of light can be adjusted to get a good contrast among test and control lines and background. In order to provide good quantitative and reproducible results, detection system should be sensitive to different intensities of colors. Optical standards can be used to calibrate an optical reader device. Automated systems have advantages over manual imaging and processing in terms of time consumption, interpretation of results and adjustment of variables.

In case of fluorescent labels, a fluorescence strip reader is used to record fluorescence intensity of test and control lines. Fluorescence brightness of test line increased with an increase in nitrated ceruloplasmin concentration in human serum when it was detected with a fluorescence strip reader. A photoelectric sensor was also used for detection in LFIA where colloidal gold is exposed to light emitting diode and resulting photoelectrons are recorded. Chemiluminescence which results from reaction of enzyme and substrate is measured as a response to amount of target analyte. Magnetic strip readers and electrochemical detectors are also reported as detection systems in LFTS but they are not very common. Selection of detector is mainly determined by the label employed in analysis.

As mentioned, the levels of TRAIL and the disease-associated parameter are combined to provide a combined score for risk analysis.

In one embodiment, when the TRAIL protein level is below a predetermined amount, the risk assessment based on the disease-associated parameter is raised. The predetermined level of serum TRAIL protein may be below 30 pg/ml, below 25 pg/ml, below 20 pg/ml, below 15 pg/ml or even below 10 pg/ml.

Exemplary ranges for indicating a high risk patient include but are not limited to 0.5-30 pg/ml, 0.5-25 pg/ml, 0.5-20 pg/ml, 0.5-15 pg/ml, 0.5-10 pg/ml, 0.5-5 pg/ml, 1-10 pg/ml; 10-20 pg/ml; 5-10 pg/ml; 5-25 pg/ml; 0.5-20 pg/ml; 0.5-5 pg/ml; 0.5-26 pg/ml; 0.5-27 pg/ml; 0.5-28 pg/ml; 0.5-29 pg/ml; 0.5-30 pg/ml; 0.5-35 pg/ml.

For example, when the level of at least one disease severity marker is indicative of a low risk patient and the TRAIL protein level is below 30 pg/ml, below 25 pg/ml, below 20 pg/ml, below 15 pg/ml or even below 10 pg/ml, the patient may be classified as an intermediate risk patient.

When the level of at least one disease severity marker is indicative of an intermediate risk patient and the TRAIL protein level is below 30 pg/ml, below 25 pg/ml, below 20 pg/ml, below 15 pg/ml or even below 10 pg/ml, the patient may be classified as a high risk patient.

When the level of at least one disease severity marker is indicative of a high risk patient and the TRAIL protein level is above 25 pg/ml, the patient may be classified as an intermediate risk patient.

For example, a patient can be considered severely ill if the APACHE II score surpasses a certain threshold and if TRAIL is below a certain threshold. In another example a patient with an APACHE II score of 0-9 will be considered as low risk if its TRAIL levels are above 25 pg/ml, but as intermediate risk if a serum TRAIL level below 25 pg/ml was detected—see for example FIG. 16C.

Below are examples of how TRAIL levels may affect the risk assessment based on individual disease severity parameters.

MR-proADM:

When used alone, levels below <0.75 nmol/L indicate low risk (e.g. outpatient care may be recommended) to patients with lower respiratory tract infections, levels between 0.75 nmol/L and 1.5 nmol/L indicate intermediate risk (e.g. short-term hospitalization may be recommended) and levels above 1.5 nmol/L indicate high risk (e.g. ICU admission may be recommended). If TRAIL levels are below 25 pg/ml, the low risk group may now be considered an intermediate or high risk group and the intermediate risk group may be considered a high risk group.

Procalcitonin:

When used alone, levels below 0.5 ng/mL indicate low risk for progression to severe sepsis and/or septic shock (e.g. outpatient care may be recommended), levels between 0.5-2 ng/ml indicate intermediate risk (e.g. short-term hospitalization may be recommended) and levels above 2.0 ng/ml indicate high risk (e.g. ICU admission may be recommended). If TRAIL levels are below 25 pg/ml, the low risk group may now be considered an intermediate or high risk group and the intermediate risk group may be considered a high risk group.

Prostate Specific Antigen:

When used alone, levels below 10 ng/mL indicate low risk (15% chance of biochemical recurrence within 5 years)—, levels between 10-20 ng/ml indicate intermediate risk (e.g. 40% chance of biochemical recurrence within 5 years), and levels above 20 ng/ml indicate high risk (e.g. 40% chance of biochemical recurrence within 5 years). If TRAIL levels are below 25 pg/ml, the low risk group may now be considered an intermediate or high risk group and the intermediate risk group may be considered a high risk group.

Troponin I:

When used alone, levels below 0.4 ng/mL indicate low risk of mortality, levels between 0.4-5 ng/ml indicate intermediate risk of mortality and levels above 5.0 ng/ml indicate high risk of mortality. If TRAIL levels are below 25 pg/ml, the low risk group may now be considered an intermediate or high risk group and the intermediate risk group may be considered a high risk group. If TRAIL levels are below 25 pg/ml, the low risk group may now be considered an intermediate or high risk group and the intermediate risk group may be considered a high risk group.

Particular combinations of TRAIL and at least one disease associated parameter are provided herein below:

TRAIL+Mid-region pro-adrenomedullin (MR-proADM)

TRAIL+CRP+ MR-proADM

TRAIL+PCT+ MR-proADM

TRAIL+IL-6+ MR-proADM

TRAIL+NGAL+ MR-proADM

TRAIL+CRP+IP-10+ MR-proADM

TRAIL+CRP+IL-6+ MR-proADM

TRAIL+PCT+IL-6+ MR-proADM

TRAIL+PCT+IP-10+ MR-proADM

TRAIL+PCT+NGAL+ MR-proADM

TRAIL+CRP+IL-6+PCT+ MR-proADM

TRAIL+CRP+IL-6+NGAL+ MR-proADM

TRAIL+CRP+IL-6+IP-10+ MR-proADM

TRAIL+NGAL+IL-6+PCT+ MR-proADM

TRAIL+IP-10+IL-6+PCT+ MR-proADM

TRAIL+WBC+ MR-proADM

TRAIL+ANC+ MR-proADM

TRAIL+WBC

TRAIL+ANC

TRAIL+temperature

TRAIL+mean arterial pressure

TRAIL+pH arterial

TRAIL+heart rate

TRAIL+respiratory rate

TRAIL+AaDO2 or PaO2

TRAIL+sodium

TRAIL+potassium

TRAIL+creatinine

TRAIL+hematocrit

TRAIL+Glasgow Coma Scale

It will be appreciated that additional markers may also be used when performing the risk analysis according to this aspect of the present invention. The additional markers may aid in increasing the accuracy of the risk analysis.

Exemplary additional markers include for example those disclosed in Table 2 herein below.

TABLE 2 RefSeq DNA Protein symbol Full Gene Name sequence RefSeq proteins CRP C-reactive protein, NC_000001.11 NP_000558.2 pentraxin-related NT_004487.20 NC_018912.2 IP-10 Chemokine (C—X—C NC_000004.12 NP_001556.2 motif) ligand 10 NC_018915.2 NT_016354.20 IL1R/IL1R1/IL1RA Interleukin 1 receptor, NC_000002.12 NP_000868.1 type I NT_005403.18 NP_001275635.1 NC_018913.2 SAA/SAA1 Serum amyloid A1 NC_000011.10 NP_000322.2 NC_018922.2 NP_001171477.1 NT_009237.19 NP_954630.1 TREM1 Triggering receptor NC_000006.12 NP_001229518.1 expressed on myeloid NT_007592.16 NP_001229519.1 cells 1 NC_018917.2 NP_061113.1 TREM2 Triggering receptor NC_000006.12 NP_001258750.1 expressed on myeloid NT_007592.16 NP_061838.1 cells 2 NC_018917.2 RSAD2 Radical S-adenosyl NC_000002.12 NP_542388.2 methionine domain NT_005334.17 containing 2 NC_018913.2 NGAL Lipocalin 2 NC_000009.12 NP_005555.2 NC_018920.2 NT_008470.20 MMP8 Matrix metallopeptidase 8 NC_000011.10 NP_001291370.1 NT_033899.9 NP_001291371.1 NC_018922.2 NP_002415.1 Procalcitonin (PCT) Calcitonin-related NC_000011.10 NP_001029124.1 polypeptide alpha NC_018922.2 NP_001029125.1 NT_009237.19 NP_001732.1 IL-6 Interleukin 6 NC_000007.14 NP_000591.1 NT_007819.18 NC_018918.2 MX1 MX Dynamin-Like NC_000021.9 NP_001138397.1 GTPase 1 NT_011512.12 NP_001171517.1 NC_018932.2 NP_001269849.1 NP_002453.2 ADM Adrenomedullin NC_000011.10 NP_001115.1 NC_018922.2 ENSP00000431438 NT_009237.19 ENSP00000433062 ENSP00000434354 ENSP00000434749 ENSP00000435124 ENSP00000436607 ENSP00000436837 ENSP00000278175 Neopterin 2-amino-6-(1,2,3- N/A N/A trihydroxypropyl)-1H- pteridin-4-one IUPAC name

Combining the levels of TRAIL and the at least one disease severity parameter (and any other contemplated marker or determinant) is typically effected using algorithms or formulas as described herein below.

A “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an “index” or “index value”. Non-limiting examples of “formulas” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value transformations and normalizations (including, without limitation, those normalization schemes based on clinical-determinants, such as gender, age, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations. Of particular use in combining determinants are linear and non-linear equations and statistical classification analyses to determine the relationship between levels of determinants detected in a subject sample and the subject's risk assessment. In panel and combination construction, of particular interest are structural and syntactic statistical classification algorithms, and methods of index construction, utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, and Hidden Markov Models, among others. Other techniques may be used in survival and time to event hazard analysis, including Cox, Weibull, Kaplan-Meier and Greenwood models well known to those of skill in the art. Many of these techniques are useful either combined with a determinant selection technique, such as forward selection, backwards selection, or stepwise selection, complete enumeration of all potential panels of a given size, genetic algorithms, or they may themselves include biomarker selection methodologies in their own technique. These may be coupled with information criteria, such as Akaike's Information Criterion (AIC) or Bayes Information Criterion (BIC), in order to quantify the tradeoff between additional biomarkers and model improvement, and to aid in minimizing overfit. The resulting predictive models may be validated in other studies, or cross-validated in the study they were originally trained in, using such techniques as Bootstrap, Leave-One-Out (LOO) and 10-Fold cross-validation (10-Fold CV).

Any formula may be used to combine TRAIL the at least one disease severity parameter results into indices useful in the practice of the invention. As indicated above, and without limitation, such indices may indicate, among the various other indications, the probability, likelihood, absolute or relative risk, time to or rate of conversion from one to another disease states, or make predictions of future biomarker measurements of the disease. This may be for a specific time period or horizon, or for remaining lifetime risk, or simply be provided as an index relative to another reference subject population.

Although various preferred formula are described here, several other model and formula types beyond those mentioned herein and in the definitions above are well known to one skilled in the art. The actual model type or formula used may itself be selected from the field of potential models based on the performance and diagnostic accuracy characteristics of its results in a training population.

Exemplary formulas include the broad class of statistical classification algorithms, and in particular the use of discriminant analysis. The goal of discriminant analysis is to predict class membership from a previously identified set of features. In the case of linear discriminant analysis (LDA), the linear combination of features is identified that maximizes the separation among groups by some criteria. Features can be identified for LDA using an eigengene based approach with different thresholds (ELDA) or a stepping algorithm based on a multivariate analysis of variance (MANOVA). Forward, backward, and stepwise algorithms can be performed that minimize the probability of no separation based on the Hotelling-Lawley statistic.

Eigengene-based Linear Discriminant Analysis (ELDA) is a feature selection technique developed by Shen et al. (2006). The formula selects features (e.g. biomarkers) in a multivariate framework using a modified eigen analysis to identify features associated with the most important eigenvectors. “Important” is defined as those eigenvectors that explain the most variance in the differences among samples that are trying to be classified relative to some threshold.

A support vector machine (SVM) is a classification formula that attempts to find a hyperplane that separates two classes. This hyperplane contains support vectors, data points that are exactly the margin distance away from the hyperplane. In the likely event that no separating hyperplane exists in the current dimensions of the data, the dimensionality is expanded greatly by projecting the data into larger dimensions by taking non-linear functions of the original variables (Venables and Ripley, 2002). Although not required, filtering of features for SVM often improves prediction. Features (e.g., biomarkers) can be identified for a support vector machine using a non-parametric Kruskal-Wallis (KW) test to select the best univariate features. A random forest (RF, Breiman, 2001) or recursive partitioning (RPART, Breiman et al., 1984) can also be used separately or in combination to identify biomarker combinations that are most important. Both KW and RF require that a number of features be selected from the total. RPART creates a single classification tree using a subset of available biomarkers.

Other formula may be used in order to pre-process the results of individual determinant measurement into more valuable forms of information, prior to their presentation to the predictive formula. Most notably, normalization of biomarker results, using either common mathematical transformations such as logarithmic or logistic functions, as normal or other distribution positions, in reference to a population's mean values, etc. are all well known to those skilled in the art. Of particular interest are a set of normalizations based on clinical-determinants such as age, time from symptoms, gender, race, or sex, where specific formula are used solely on subjects within a class or continuously combining a clinical-determinants as an input. In other cases, analyte-based biomarkers can be combined into calculated variables which are subsequently presented to a formula.

In addition to the individual parameter values of one subject potentially being normalized, an overall predictive formula for all subjects, or any known class of subjects, may itself be recalibrated or otherwise adjusted based on adjustment for a population's expected prevalence and mean biomarker parameter values, according to the technique outlined in D'Agostino et al, (2001) JAMA 286:180-187, or other similar normalization and recalibration techniques. Such epidemiological adjustment statistics may be captured, confirmed, improved and updated continuously through a registry of past data presented to the model, which may be machine readable or otherwise, or occasionally through the retrospective query of stored samples or reference to historical studies of such parameters and statistics. Additional examples that may be the subject of formula recalibration or other adjustments include statistics used in studies by Pepe, M. S. et al, 2004 on the limitations of odds ratios; Cook, N. R., 2007 relating to ROC curves. Finally, the numeric result of a classifier formula itself may be transformed post-processing by its reference to an actual clinical population and study results and observed endpoints, in order to calibrate to absolute risk and provide confidence intervals for varying numeric results of the classifier or risk formula.

Some determinants may exhibit trends that depends on the patient age (e.g. the population baseline may rise or fall as a function of age). One can use an ‘Age dependent normalization or stratification’ scheme to adjust for age related differences. Performing age dependent normalization or stratification can be used to improve the accuracy of determinants for differentiating between different types of infections. For example, one skilled in the art can generate a function that fits the population mean levels of each determinant as function of age and use it to normalize the determinant of individual subjects levels across different ages. Another example is to stratify subjects according to their age and determine age specific thresholds or index values for each age group independently.

As mentioned the level of TRAIL can be used to make a risk assessment as to a patient and further to decide on the appropriate treatment course.

Thus, according to another aspect of the present invention there is provided a method of determining a treatment course for a subject presenting with an appendicitis comprising measuring the TRAIL protein level in a blood sample of the subject, wherein when the level is below a predetermined amount the subject is recommended for appendectomy.

Typically the subject presenting with the appendicitis exhibits at least one, two, three, four or all of the following symptoms: elevated CRP levels, PCT levels, findings on ultrasound or CT scan, nausea and vomiting, epigastric pain migrating to the right lower quadrant (RLQ), tenderness in RLQ, rebound pain, elevated temperature, shift of WBCs to the left, anorexia or leukocytosis.

Measuring the TRAIL protein level has been described herein above.

According to this aspect of the present invention, the predetermined amount below which the subject is recommended for appendectomy is below 30 pg/ml, below 25 pg/ml, below 20 pg/ml, below 15 pg/ml or even below 10 pg/ml. Exemplary ranges for appendectomy treatment include but are not limited to 0.5-30 pg/ml, 0.5-25 pg/ml, 0.5-20 pg/ml, 0.5-15 pg/ml, 0.5-10 pg/ml, 0.5-5 pg/ml.

Conversely, when the TRAIL level is above this predetermined amount, the subject may be recommended non-surgical treatments—e.g. antibiotic treatment. In one embodiment, the when the level is between 30-70 pg/ml, the subject is recommended a non-surgical treatment, when the level is between 25-70 pg/ml, the subject is recommended a non-surgical treatment, when the level is between 30-80 pg/ml, the subject is recommended a non-surgical treatment, when the level is between 25-80 pg/ml, the subject is recommended a non-surgical treatment, when the level is between 20-70 pg/ml, the subject is recommended a non-surgical treatment, when the level is between 20-80 pg/ml, the subject is recommended a non-surgical treatment.

In one embodiment, the TRAIL levels are used to improve the accuracy of various scoring systems aimed to identify acute appendicitis or to stratify acute appendicitis patients according to disease severity in order to recommend appendectomy or antibiotic treatment. An example of such scoring system is the MANTRELS score.

As well as using the TRAIL protein serum levels, the present inventors contemplate use of additional factors and measurements to help decide a treatment course for the subject with appendicitis. Such factors include, but are not limited to white blood cell count (WBC) and C-reactive protein (CRP) blood level. Additional markers that may be used for determining treatment for appendicitis are any of those described herein (see for example Table 2).

The present inventors have further shown that patients with TRAIL levels below 25 pg/ml were more likely to have serious bacterial infections, (e.g. invasive bacterial infections) or other severe clinical syndromes, for example bacteremia and septic shock, as these syndromes were statistically enriched in the low TRAIL sub-group (64% (7/11) of all bacteremia cases P<10⁻³, and 100% (7/7) of all septic shock cases, P<10⁻⁶; FIG. 15.

Thus, according to yet another aspect of the present invention there is provided a method of diagnosing a serious bacterial infection in a subject comprising measuring the TRAIL protein level in a blood sample of the subject, wherein when the TRAIL protein level is below 25 pg/ml it is indicative of a serious bacterial infection.

The method according to this aspect of the present invention may be used to “rule in” a serious bacterial infection. Alternatively, the method may be used to rule out a non-serious bacterial infection. The method according to some embodiments can be used to “rule in” a serious bacterial infection and “rule out” a non-bacterial disease.

Serious bacterial infections may include for example the following clinical syndromes: meningitis, sepsis, pneumonia, septic arthritis and cellulitis, bacteremia, urinary tract infection (UTI), and Pyelonephritis.

According to a particular embodiment, the serious bacterial infection is not sepsis.

Thus, according to yet another aspect of the present invention there is provided a method of diagnosing an invasive bacterial infection in a subject comprising measuring the TRAIL protein level in a blood sample of the subject, wherein when the TRAIL protein level is below 25 pg/ml it is indicative of an invasive bacterial infection.

The method according to this aspect of the present invention may be used to “rule in” an invasive bacterial infection. Alternatively, the method may be used to rule out a non-invasive bacterial infection. The method according to some embodiments can be used to “rule in” an invasive bacterial infection and “rule out” a non-bacterial disease.

Invasive bacterial infections occur when the bacteria get past the defenses of the person who is infected. This may occur when a person has sores or other breaks in the skin that allow the bacteria to get into the tissue, or when the person's ability to fight off the infection is decreased because of chronic illness or an illness that affects the immune system.

Although healthy people can get invasive bacterial disease, people with chronic illnesses like cancer, diabetes, and chronic heart or lung disease, and those who use medications such as steroids have a higher risk. Persons with skin lesions (such as cuts, chicken pox, surgical wounds), the elderly, and adults with a history of alcohol abuse or injection drug use also have a higher risk for disease.

Exemplary invasive bacterial diseases include but are not limited to Meningococcal Disease, Invasive; Staphylococcus Aureus Infections; Drug resistant (MRSA, VISA, VRSA) Streptococcal Disease, Group A Invasive or Streptococcal TSS; Streptococcal Disease, Invasive Group B.

Other contemplated invasive bacterial diseases include bacteremia, septic arthritis, abdominal abscess, bacterial meningitis, mastoiditis, nephronia, peritonsillar abscess, deep neck infection, periappendicular abscess and septic shock.

According to a particular embodiment, the invasive bacterial disease is not septic shock.

It will be appreciated that as well as measuring TRAIL, the present inventors contemplate use of additional markers that may aid in the diagnosis of invasive bacterial disease. These include any of the markers taught in the present application such as procalcitonin, lactate, lactic acid, CRP, WBC, ANC, or pathogen related determinants (such as nucleic acids) of one of the following pathogens: Staphylococcus Aureus; Drug resistant Staphylococcus Aureus (MRSA, VISA, VRSA), Group A or Group B Streptococci.

Measuring serum protein TRAIL levels is described herein above.

In one embodiment, when the TRAIL protein level is below a predetermined amount, the chance that the subject is suffering from an invasive bacterial infection or from a serious bacterial infection is increased. The predetermined level of serum TRAIL protein may be below 30 pg/ml, below 25 pg/ml, below 20 pg/ml, below 15 pg/ml or even below 10 pg/ml.

Exemplary ranges for indicating a high risk of invasive bacterial infection include but are not limited to 0.5-30 pg/ml, 0.5-25 pg/ml, 0.5-20 pg/ml, 0.5-15 pg/ml, 0.5-10 pg/ml, 0.5-5 pg/ml, 1-10 pg/ml; 10-20 pg/ml; 5-10 pg/ml; 5-25 pg/ml; 0.5-20 pg/ml; 0.5-5 pg/ml; 0.5-26 pg/ml; 0.5-27 pg/ml; 0.5-28 pg/ml; 0.5-29 pg/ml; 0.5-30 pg/ml; 0.5-35 pg/ml.

In the context of the present invention, the following abbreviations may be used: ANC=Absolute neutrophil count; ANN=Artificial neural networks; AUC=Area under the receiver operating curve; BP=Bordetella pertussis; CHF=Congestive heart failure; CI=Confidence interval; CID=Congenital immune deficiency; CLL=Chronic lymphocytic leukemia; CMV=Cytomegalovirus; CNS=Central nervous system; COPD=Chronic obstructive pulmonary disease; CP=Chlamydophila pneumonia; CRP=C-reactive protein; CSF=Cerebrospinal fluid; CV=Coefficient of variation; DOR=Diagnostic odds ratio; EBV=Epstein bar virus; eCRF=Electronic case report form; ED=Emergency department, ELISA=Enzyme-linked immunosorbent assay; FDR=False discovery rate; FMF=Familial Mediterranean fever; G-CSF=Granulocyte colony-stimulating factor; GM-CSF=Granulocyte-macrophage colony-stimulating factor; HBV=Hepatitis B virus; HCV=Hepatitis C virus; HI=Haemophilus influenza; HIV=Human immunodeficiency virus; IDE=Infectious disease experts; IL=Interleukin; IRB=institutional review board; IVIG=Intravenous immunoglobulin; KNN=K-nearest neighbors; LP=Legionella pneumophila; LR+=Positive likelihood ratio; LR-=Negative likelihood ratio; LRTI=Lower respiratory tract infections; mAb=Monoclonal antibodies; MDD=Minimum detectable dose; MDS=Myelodysplastic syndrome; MP=Mycoplasma pneumonia; MPD=Myeloproliferative disease; NPV=Negative predictive value; PCT=Procalcitonin; PED=Pediatric emergency department; PPV=Positive predictive value; QA=Quality assurance; RSV=Respiratory syncytial virus; RV=Rhinovirus; SIRS=systemic inflammatory syndrome; SP=Streptococcus pneumonia; STARD=Standards for Reporting of Diagnostic Accuracy; SVM=Support vector machine; TNF=Tumor necrosis factor; URTI=Upper respiratory tract infection; UTI=Urinary tract infection; WBC=White blood cell; WS=Wilcoxon ranksum.

In the context of the present invention, the following statistical terms may be used:

“TP” is true positive, means positive test result that accurately reflects the tested-for activity. For example in the context of the present invention a TP, is for example but not limited to, truly classifying a bacterial infection as such.

“TN” is true negative, means negative test result that accurately reflects the tested-for activity. For example in the context of the present invention a TN, is for example but not limited to, truly classifying a viral infection as such.

“FN” is false negative, means a result that appears negative but fails to reveal a situation. For example in the context of the present invention a FN, is for example but not limited to, falsely classifying a bacterial infection as a viral infection.

“FP” is false positive, means test result that is erroneously classified in a positive category. For example in the context of the present invention a FP, is for example but not limited to, falsely classifying a viral infection as a bacterial infection.

“Sensitivity” is calculated by TP/(TP+FN) or the true positive fraction of disease subjects.

“Specificity” is calculated by TN/(TN+FP) or the true negative fraction of non-disease or normal subjects.

“Total accuracy” is calculated by (TN+TP)/(TN+FP+TP+FN).

“Positive predictive value” or “PPV” is calculated by TP/(TP+FP) or the true positive fraction of all positive test results. It is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested.

“Negative predictive value” or “NPV” is calculated by TN/(TN+FN) or the true negative fraction of all negative test results. It also is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested. See, e.g., O'Marcaigh A S, Jacobson R M, “Estimating The Predictive Value Of A Diagnostic Test, How To Prevent Misleading Or Confusing Results,” Clin. Ped. 1993, 32(8): 485-491, which discusses specificity, sensitivity, and positive and negative predictive values of a test, e.g., a clinical diagnostic test.

“MCC” (Mathews Correlation coefficient) is calculated as follows: MCC=(TP*TN−FP*FN)/{(TP+FN)*(TP+FP)*(TN+FP)*(TN+FN)}{circumflex over ( )}0.5 where TP, FP, TN, FN are true-positives, false-positives, true-negatives, and false-negatives, respectively. Note that MCC values range between −1 to +1, indicating completely wrong and perfect classification, respectively. An MCC of 0 indicates random classification. MCC has been shown to be a useful for combining sensitivity and specificity into a single metric (Baldi, Brunak et al. 2000). It is also useful for measuring and optimizing classification accuracy in cases of unbalanced class sizes (Baldi, Brunak et al. 2000).

“Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value. Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus misclassified outcomes (false positives (FP) or false negatives (FN)), and may be stated as a sensitivity, specificity, positive predictive values (PPV) or negative predictive values (NPV), Mathews correlation coefficient (MCC), or as a likelihood, odds ratio, Receiver Operating Characteristic (ROC) curve, Area Under the Curve (AUC) among other measures.

“Analytical accuracy” refers to the reproducibility and predictability of the measurement process itself, and may be summarized in such measurements as coefficients of variation (CV), Pearson correlation, and tests of concordance and calibration of the same samples or controls with different times, users, equipment and/or reagents. These and other considerations in evaluating new biomarkers are also summarized in Vasan, 2006.

“Performance” is a term that relates to the overall usefulness and quality of a diagnostic or prognostic test, including, among others, clinical and analytical accuracy, other analytical and process characteristics, such as use characteristics (e.g., stability, ease of use), health economic value, and relative costs of components of the test. Any of these factors may be the source of superior performance and thus usefulness of the test, and may be measured by appropriate “performance metrics,” such as AUC and MCC, time to result, shelf life, etc. as relevant.

By “statistically significant”, it is meant that the alteration is greater than what might be expected to happen by chance alone (which could be a “false positive”). Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is often considered highly significant at a p-value of 0.05 or less.

The term “determinant” as used herein refers to a disease associated parameter or biomarker.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Example 1

Methods

Study Population:

1002 patients with suspected acute infectious disease and non-infectious controls were prospectively recruited from Hillel-Yaffe and Bnai-Zion Medical Centers, Israel (NCT01917461). The study was conducted according to the guidelines and recommendations of Good Clinical Practice and the Declaration of Helsinki. Written informed consent was obtained from each participant or legal guardian, as applicable.

Pediatric patients (≤18 years) were recruited from pediatric emergency departments (PED), pediatric wards and surgical departments, and adults (>18 years) from emergency departments (ED), internal medicine departments and surgical departments. Informed consent was obtained from each participant or legal guardian, as applicable. Inclusion criteria for the infectious disease cohort included: clinical suspicion of an acute infectious disease, peak fever >37.5° C. since symptoms onset, and duration of symptoms ≤12 days. Inclusion criteria for the control group included: clinical impression of a non-infectious disease (e.g. trauma, stroke and myocardial infarction), or healthy subjects. Exclusion criteria included: evidence of any episode of acute infectious disease in the two weeks preceding enrollment; diagnosed congenital immune deficiency; current treatment with immunosuppressive or immunomodulatory therapy; active malignancy, proven or suspected human immunodeficiency virus (HIV)-1, hepatitis B virus (HBV), or hepatitis C virus (HCV) infection. Importantly, in order to enable broad generalization, antibiotic treatment at enrollment did not cause exclusion from the study.

Enrollment Process and Data Collection:

For each patient, the following baseline variables were recorded: demographics, physical examination, medical history (e.g. main complaints, underlying diseases, chronically-administered medications, comorbidities, time of symptom onset, and peak temperature), complete blood count (CBC) obtained at enrollment, and chemistry panel (e.g. creatinine, urea, electrolytes, and liver enzymes). A nasal swab was obtained from each patient for further microbiological investigation, and a blood sample was obtained for protein screening and validation. Additional samples were obtained as deemed appropriate by the physician (e.g. urine and stool samples in cases of suspected urinary tract infection [UTI], and gastroenteritis [GI] respectively). Radiological tests were obtained at the discretion of the physician (e.g. chest X-ray for suspected lower respiratory tract infection [LRTI]). Thirty days after enrollment, disease course and response to treatment were recorded. All information was recorded in a custom electronic case report form (eCRF).

Microbiological Investigation:

Patients underwent two multiplex-PCR diagnostic assays from nasal swab samples: (i) Seeplex™ RV15 (n=713), for detection of parainfluenza virus 1, 2, 3, and 4, coronavirus 229E/NL63, adenovirus A/B/C/D/E, bocavirus 1/2/3/4, influenza virus A and B, metapneumovirus, coronavirus OC43, rhinovirus A/B/C, respiratory syncytial virus A and B, and Enterovirus, and (ii) Seeplex™ PB6 (n=633) for detection of Streptococcus pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Legionella pneumophila, Bordetella pertussis, and Mycoplasma pneumoniae. Multiplex-PCR assays were performed by a certified service laboratory. Patients were also tested for additional pathogens according to their suspected clinical syndrome, including: blood culture (n=420), urine culture (n=188) and stool culture for Shigella spp., Campylobacter spp. and Salmonella spp. (n=66); serological testing (IgM and/or IgG) for cytomegalovirus (CMV), Epstein-Barr virus (EBV), Mycoplasma Pneumonia, and Coxiella burnetii (Q-Fever) (n=167, n=130, n=206 and n=41 respectively).

Establishing the Reference Standard:

A rigorous composite reference standard was created following recommendations of the Standards for Reporting of Diagnostic Accuracy (STARD; FIG. 1).¹ First, a thorough clinical and microbiological investigation was performed for each patient as described above. Then, all the data collected throughout the disease course was reviewed by a panel of three physicians. For adult patients (>18 years) the panel included the attending physician and two infectious disease specialists, while for children and adolescents (≤18 years) it included the attending pediatrician, an infectious disease expert and a senior attending pediatrician. Each panel member assigned one of the following diagnostic labels to each patient: (i) bacterial; (ii) viral; (iii) no apparent infectious disease or healthy (controls); and (iv) mixed infections (bacteria plus virus). Importantly, the panel members were blinded to the labeling of their peers and to the results of the signature.

Patient Prognostic Measures:

Various clinical measures were used to retrospectively asses patient's prognosis such as ICU admission, need for mechanical ventilation or surgical interventions, hospital length of stay, patient re-admission to the hospital, and the manifestation of a severe clinical syndrome like bacteremia or septic shock.

Samples, Procedures and Protein Measurements:

Venous blood samples were stored at 4° C. for up to 5 hours on site and subsequently fractionated into plasma, serum and total leukocytes and stored at −80° C. Nasal swabs and stool samples were stored at 4° C. for up to 72 hours and subsequently transported to a certified service laboratory for multiplex PCR-based assay. TRAIL was measured using commercial ELISA kits (MeMed Diagnostics).

Statistical Analysis:

The primary analysis was based on area under the receiver operating characteristics curve (AUC), Sensitivity (TP/P), Specificity (TN/N), Positive predictive value (PPV=TP/[TP+FP]), Negative predictive value (NPV=TN/[TN+FN]), where P, N, TP and TN correspond to positives (bacterial patients), negatives (viral patients), true positives (correctly diagnosed bacterial patients), and true negatives (correctly diagnosed viral patients), respectively. Statistical analysis was performed with MATLAB.

Results

Patient Characteristics:

Three physicians independently assigned a label to each patient (either bacterial, viral, controls, or indeterminate). 98 patients were labeled as indeterminate, because the physicians could not establish disease etiology or there was no majority labeling. A detailed characterization of the analyzed cohort is depicted in FIGS. 2-8. Briefly, the cohort was balanced with respect to gender (47% females, 53% males) and included 56% pediatric patients (≤18 years) and 44% adults (>18 years). Patients presented with a wide range of clinical syndromes (e.g. RTI, UTI, and systemic infections), maximal temperatures (36-41.5° C.), and time from symptoms onset (0-12 days). Altogether, 56 pathogen species were detected that are responsible for the vast majority of acute infectious diseases in the Western world.

TRAIL as a Marker for Predicting Disease Severity and Risk Assessment:

In the studied cohort, 667 patients had TRAIL levels higher than 25 pg/ml and 98 patients had TRAIL levels lower than 25 pg/ml (FIG. 11), out of which 89 had a bacterial etiology, 4 viral etiology and 5 with non-infectious condition. The 98 patients with TRAIL levels lower than 25 pg/ml had poorer patient prognosis and outcome, and higher disease severity compared to patients with higher TRAIL levels. For example, their hospitalization duration was close to 4-fold longer compared to patients with higher TRAIL levels (7.5±1.17 vs. 1.9±0.1, days, average±standard error, P<10⁻⁵; FIG. 12). All patients from the infectious disease group that required mechanical ventilation and ICU admission had TRAIL levels lower than 25 pg/ml (6/93 TRAIL <25 pg/ml vs. 0/560 TRAIL >25 pg/ml, P<10⁻⁵; FIG. 13). Median serum concentrations were 9 pg/ml vs. 80 pg respectively, (ranksum P<0.001, FIG. 14), for severely ill and all other patients respectively. Strikingly, the lowest TRAIL levels (<5 pg/ml) were measured in the only two children that died in the entire cohort. Finally, patients with TRAIL levels <25 pg/ml were more likely to have invasive bacterial infections or other severe clinical syndromes, for example bacteremia and septic shock, as these syndromes were statistically enriched in the low TRAIL sub-group (64% (7/11) of all bacteremia cases P<10⁻³, and 100% (7/7) of all septic shock cases, P<10⁻⁶; (FIG. 15).

Example 2

Appendicitis is defined as an inflammation of the inner lining of the vermiform appendix that spreads to its other parts. Despite diagnostic and therapeutic advancement in medicine, appendicitis remains a clinical emergency and is one of the more common causes of acute abdominal pain. Laboratory tests such as CBC, CRP, liver, pancreatic function tests and urine analysis do not have findings specific for appendicitis, but they may be helpful to confirm diagnosis in patients with an atypical presentation. Appendectomy remains the standard treatment of appendicitis, however, in the last few years a number of studies regarding an alternative, antibiotic approach, have been conducted. Preliminary evidence suggests that non-operative management in selected low risk patients with acute appendicitis exhibited higher quality of life scores and is associated with lower appendicitis-related health care costs [Minneci P C, et al J Am Coll Surg 2014; 219:272; Svensson J F, et al. Ann Surg 2015; 261:67; Minneci P C, et al. JAMA Surg 2016; 151:408; Telem D A. JAMA 2016; 315:811; Tanaka Y, et al. J Pediatr Surg 2015; 50:1893]. Furthermore, early detection and treatment of patients who are at high risk of appendicitis complications, such as perforation and peritonitis, is imperative and should promote early appendectomy[Bickell N A, J Am Coll Surg 2006; 202:401; Nomura O, et al. Pediatr Emerg Care 2012; 28:792].

Thus new diagnostic and prognostic markers are needed to successfully differentiate patients who will benefit an operative management from those who will be better treated with a more conservative approach. The present inventors therefore sought to evaluate the ability of TRAIL to predict outcome and recommend proper management course in acute appendicitis patients.

Methods

Presented herein, are two case studies of acute appendicitis patients with different disease course for whom blood TRAIL levels were measured. Venous blood samples were stored at 4° C. for up to 5 hours on site and subsequently fractionated into plasma, serum and total leukocytes and stored at −80° C. TRAIL was measured from serum fraction using commercial ELISA kits (MeMed Diagnostics).

Results

Case Number 10542

A 13 year old male presented to the emergency department with a two day history of fever, abdominal pain and vomiting. On physical examination, RLQ tenderness with positive rebound and Mcburney signs were noted.

Main laboratory results: CRP 396.5 mg/1, WBC 17,090 cells/μl

Ultrasound test revealed an inflamed appendix, 10 mm in diameter and extensive gutters fluid collection. An early appendectomy operation was conducted in which a perforated appendix was noticed and resected. On the 6^(th) post-operative day surgical wound infection was noted with purulent discharge, on ultrasound exam 6 cm pelvic fluid collection. Compatible with wound culture results, patient received I.V Ceftriaxone and Metronidazole. Patient was discharged after a total hospitalization duration of 21 days.

Measured TRAIL levels: 15.4 pg/ml.

Case Number: 10565

A three-year-old boy presented to the emergency department with a four day history of fever up to 41.4° C. (106.5° F.), chills and emesis a day prior to presentation. On physical examination, the patient appeared sick, showed right lower quadrant (RLQ) tenderness and had a positive rebound sign without peritonitis. Main laboratory results: CRP 242.2 mg/1, WBC 13,000 cells/W. On ultrasound test, the appendix could not be visualized. The patient was scheduled for an early appendectomy operation, however, due to parents' objection to an operation and a CT scan, conservative antibiotic treatment with intravenous (I.V) Ceftriaxone was initiated. A second ultrasound test, a day following antibiotic treatment administration, revealed normal appendix without signs of inflammation. Patient was discharged without surgical procedure.

Measured TRAIL levels: 80.9 pg/ml.

In case #10565, TRAIL levels were in the normal range (FIG. 10) and correctly predicted a positive outcome using a conservative antibiotic treatment. Low TRAIL levels were a successful indicator of a severe disease state of a perforated appendix in case #10542. These two cases demonstrate the ability of TRAIL to differentiate between mild and severe cases of suspected appendicitis and to recommend either conservative drug treatment vs invasive operative treatment according to TRAIL levels.

REFERENCES

-   1. Bossuyt, P. M. et al. The STARD Statement for Reporting Studies     of Diagnostic Accuracy: Explanation and Elaboration. Ann Intern Med     138, W1-W12 (2003). -   2. Niemz, A., Ferguson, T. M. & Boyle, D. S. Point-of-care nucleic     acid testing for infectious diseases. Trends Biotechnol. 29, 240-250     (2011). -   3. Craw, P. & Balachandran, W. Isothermal nucleic acid amplification     technologies for point-of-care diagnostics: a critical review. Lab     Chip 12, 2469-2486 (2012). -   4. Kim, K. H., Shin, J. H. & Kim, S. Y. The Clinical Significance of     Nasopharyngeal Carriages in Immunocompromised Children as Assessed.     The Korean Journal of Hematology 44, 220 (2009). -   5. Shin, J. H., Han, H. Y. & Kim, S. Y. Detection of nasopharyngeal     carriages in children by multiplex reverse transcriptase-polymerase     chain reaction. Korean Journal of Pediatrics 52, 1358 (2009). -   6. Jung, C. L., Lee, M. A. & Chung, W. S. Clinical Evaluation of the     Multiplex PCR Assay for the Detection of Bacterial Pathogens in     Respiratory Specimens from Patients with Pneumonia. Korean Journal     of Clinical Microbiology 13, 40 (2010). -   7. Rhedin, S. et al. Clinical Utility of PCR for Common Viruses in     Acute Respiratory Illness. Pediatrics peds. 2013-3042 (2014).     doi:10.1542/peds.2013-3042 -   8. Bogaert, D., De Groot, R. & Hermans, P. W. M. Streptococcus     pneumoniae colonisation: the key to pneumococcal disease. Lancet     Infect Dis 4, 144-154 (2004). -   9. Spuesens, E. B. M. et al. Carriage of Mycoplasma pneumoniae in     the Upper Respiratory Tract of Symptomatic and Asymptomatic     Children: An Observational Study. PLoS Med 10, e1001444 (2013). 

What is claimed is:
 1. A method of treating a subject presenting with an appendicitis comprising: (a) measuring TNF-related apoptosis-inducing ligand (TRAIL) protein level in a blood sample of the subject; and (b) performing an appendectomy when said level is below 25 pg/ml, wherein the subject is under 18 years old.
 2. The method of claim 1, wherein when said level is above a predetermined amount the subject is recommended for non-surgical treatment.
 3. The method of claim 2, wherein said non-surgical treatment comprises antibiotic treatment.
 4. The method of claim 1, further comprising measuring white blood cell count (WBC) and/or C-reactive protein (CRP).
 5. The method of claim 1, wherein said blood sample is a fraction of whole blood.
 6. The method of claim 5, wherein said fraction is serum or plasma.
 7. The method of claim 1, wherein said blood sample comprises cells selected from the group consisting of lymphocytes, monocytes and granulocytes.
 8. The method of claim 1, wherein said measuring is determined electrophoretically or immunochemically.
 9. The method of claim 8, wherein said immunochemical determination is effected by flow cytometry, radioimmunoas say, immunofluorescence, lateral flow immunoassay or by an enzyme-linked immunosorbent assay. 